These days, structural biologists are revealing the intricate shapes of proteins  nature's own nanomachines  at a prodigious rate. Now generating about 800 structures per year, the process has become sufficiently fast enough that this past summer the NIH launched the second phase of the Protein Structure Initiative, an ambitious plan to uncover 5,000 more protein structures within the next five years.

But obtaining structures hasn't always been so efficient. One of the first protein structures solved with X-ray crystallography  the method used to "see" molecules  took 22 years of painstaking work until the final shape took form in the 1960s. Even as recently as 1990, the process could still take several years and the number of structures known at that time still only numbered in the hundreds.

And then Wayne Hendrickson University Professor of Biochemistry and Molecular Biophysics, went MAD.

MAD  "multiple-wavelength anomalous diffraction"  is an X-ray crystallography technique Dr. Hendrickson developed that removed the major bottleneck in biological structure determination.

"We chose the acronym with reference to the alternate MIR (multiple isomorphus replacement) method, but it was also catchy," Dr. Hendrickson says.

Proteins & Drug Development

Wayne Hendrickson's work on protein structure is critical to the effort to find new drugs for many diseases.

The New York Structural Biology Center, a consortium of 10 biomedical research centers in New York State, has received a five-year, $17.6 million NIH grant for a specialized center, led by Dr. Hendrickson, called the New York Consortium on Membrane Protein Structure. The consortium will focus on a key class of proteins that serve as portals through which cells and some components within cells communicate with the outside environment. Malfunctioning proteins often lead to disease and are therefore prime targets for the development of drugs.

"Many of us believe that the development of therapeutics based on a protein's structure is a much more efficient way to find the drugs and vaccines we'd like to have," Dr. Hendrickson says.

What used to be a monumental effort to determine a structure is now, because of MAD and increased computing power only a matter of a few weeks or months worth of work. Dan Leahy, professor of biophysics and biophysical chemistry at Johns Hopkins, and a former Hendrickson postdoc says, "Without MAD, the Protein Structure Initiative wouldn't even be conceivable."

In X-ray crystallography, biologists first crystallize a protein and then bombard the crystals with X-rays. When the X-rays hit the crystallized proteins they bounce off in different directions, like sunlight scattered around a room by a chandelier. The pattern of diffracted X-rays combined with a lot of mathematics is used to reconstruct  or "solve"  the structure of the molecule.

But X-ray diffraction patterns alone do not give structural biologists all the information they need to solve the structure; two types of information are required from the diffracted X-rays: their amplitudes and their phases. Amplitude is easy to measure, but phase  the position of the peaks and troughs of the wave relative to some origin  can't be detected.

"We have to play tricks to tease out the phase information, and the standard trick before MAD was to repeat the process after replacing one of the atoms in the molecule with a heavy atom, which generates a slightly different diffraction pattern," Dr. Leahy says. "By comparing those patterns we could determine the phases."

"First you'd collect data on a crystal, add mercury with the goal of having it bind in a defined place on each protein molecule in the crystal. You'd then collect more data on this mercury derivative, and do it all over again with platinum and other heavy atoms," says Larry Shapiro, Ph.D., associate professor of biochemistry and molecular biophysics. "This required lots of chemistry to obtain the same shaped, or isomorphous, crystals every time. The only difference in these multiple data sets should come from the addition of a few heavy atoms. There were problems with this method and you could get stuck for years in the chemistry phase."

When Dr. Hendrickson arrived at Columbia in 1984, he began to think the phase information could be obtained differently using a new source of X-rays produced by synchotrons, football field-sized particle accelerators used mostly by physicists and material engineers. The advantage of synchotron X-rays over sources available in the lab, Dr. Hendrickson says, is that they can be tuned to different wavelengths. By changing the wavelength of the X-rays bombarding a crystal, different diffraction patterns  and thus phase information  are generated from a single crystal.

"The work of MAD occurred over a span of several years, and at first many people felt it was experimentally too difficult to apply on a widespread basis," Dr. Leahy says. "But after first showing it was possible, Dr. Hendrickson then proved it was the easiest way. It works so well that the need to develop new phasing techniques isn't very great."

"I think of Wayne as one of the fathers of modern biological X-ray crystallography," says Dr. Shapiro. "He wasn't the only person to say MAD could work in theory, but he was the first to show it worked in reality, and he turned it into the workhorse of structural biology. The MAD technique has become the standard method for solving unknown structures."